Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of Rat Retinal Ganglion Cell Reaggregates and Optic Nerve Oligodendrocyte Precursor Cells Trent A. Watkins and Anja R. Scholze Cold Spring Harb Protoc; doi: 10.1101/pdb.prot074971 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Immunoaffinity Purification (34 articles) Immunoseparation (21 articles) Mouse (307 articles) Neural Cell Culture (52 articles) Neuroscience, general (255 articles) Other Laboratory Organisms (56 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Myelinating Cocultures of Rat Retinal Ganglion Cell Reaggregates and Optic Nerve Oligodendrocyte Precursor Cells Trent A. Watkins1,2,3 and Anja R. Scholze1 1
Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125
This protocol describes the generation of a rapidly myelinating central nervous system coculture for the study of complex neuronal-glial interactions in vitro. Postnatal rat retinal ganglion cells (RGCs) purified by immunopanning are promoted to cluster into reaggregates and then allowed to extend dense beds of radial axons for 10–14 d. Subsequently, rodent oligodendrocyte precursor cells are purified by immunopanning, transfected if desired, and seeded on top of the RGC reaggregates. Under the conditions described here, compact myelin can be observed within 6 d.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
BSA stock (4%) DNase To prepare a 0.4% stock of DNase in Earle’s balanced salt solution (EBSS), add 1 mL of Earle’s balanced salt solution (EBSS; Sigma-Aldrich E6267) per 12,500 U of DNase I (Worthington LS002007). Keep on ice. Filter sterilize, and store in 200-μL aliquots at −20˚C.
Dulbecco’s phosphate-buffered saline (D-PBS, Gibco 14287-080) Earle’s balanced salt solution (EBSS) (Sigma-Aldrich E6267) Fetal calf serum (FCS) (Gibco 10437-028) Prepare 50-mL aliquots of FCS. Heat-inactivate aliquots for 30 min at 55˚C, and then store at −20˚C.
High-ovomucoid (high-ovo) stock solution (6×) Immunopanning reagents A2B5 hybridoma supernatant or ascites Galactocerebroside (GC) hybridoma supernatant Goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch 115-005-044)
2
Present address: Department of Neuroscience, Genentech Inc., South San Francisco, California 94080. Correspondence: [email protected]
3
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1080
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
Goat anti-mouse IgM, μ-chain specific (Jackson ImmunoResearch 115-005-020) Ran-2 hybridoma supernatant
InSolution γ-Secretase Inhibitor I× (DAPT) (Calbiochem/EMD Millipore 565784) (optional; see Step 47) Insulin stock (0.5 mg/mL) L-Cysteine hydrochloride monohydrate (Sigma-Aldrich C7880) Laminin (mouse) (Cultrex; R&D Systems 3400-010-01) Thaw mouse laminin (1 mg/mL) at 4˚C. Make 10-µL aliquots and store at −30˚C.
Low-ovomucoid (low-ovo) stock solution Myelination medium (MyM), freshly prepared NaOH (1 M) ND-SATO growth medium (ND-G), freshly prepared Neurobasal solution (NB) (Gibco 21103-049) Nucleofector kit for rat glial cells (Lonza) (if performing transfection) Papain (Worthington LS03126) Plasmid or siRNA for transfection, as desired Poly-D-lysine (PDL) (1 mg/mL) Rat pups (P5, Sprague–Dawley) for purification of RGCs (three litters/30 animals) P4–P10 pups may be used, but P5 is optimal for its combination of yield, viability, and purity. RGCs are purified using the method described in Purification and Culture of Retinal Ganglion Cells from Rodents (Winzeler and Wang 2013). Mouse RGCs can also be used, per the provided modifications.
Rat pups (P7, Sprague–Dawley) for purification of optic nerve OPCs (3 litters/30 animals) Pups of nearly any postnatal age will work for OPC purification, but P6–P8 produces the best yield. Cortical OPCs can also be used in the myelinating cocultures and the OPC yields are much higher (2–3 million per P7 cortex; see Purification of Oligodendrocyte Precursor Cells from Rat Cortices by Immunopanning [Dugas and Emery 2013]). Mouse OPCs can also be used, per the provided modifications.
Tris-HCl (50 mM, pH 9.5; sterile) Dissolve 12.1 g of Trizma base in 200 mL of dH2O. Adjust pH to 9.5 with HCl.
Trypsin stock (2.5%) Dissolve trypsin (Sigma-Aldrich T9935) at 30,000 U/mL in EBSS (Sigma-Aldrich E6267). Filter through a 0.22-µm filter. Make 200-µL aliquots and store at −80˚C.
Trypsin-EDTA (Gibco) (if performing transfection) Equipment
Centrifuge (tabletop, with 15- and 50-mL conical tube adaptors) Centrifuge (clinical) Chamber slides (eight well) Coverslips (glass) We wash our glass coverslips with at least three washes of 70% ethanol over 7 d on a rotating platform and store in 70% ethanol.
Dissection tools Forceps Scissors (fine) Scissors (sharp)
Incubator (37˚C/10% CO2) Microscope Needles (#21 and #23 gauge) Nylon mesh filter (20 µm) (Nitex; Tetko) Nucleofector I device (Lonza) (if performing transfection) Pasteur pipettes Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1081
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
Petri dishes (35, 10, 15 cm) Syringes (2.5 mL) Syringe filters (0.22 µm) Tissue culture dishes (10 cm) (if performing transfection) Tissue culture plates (24-well) Tubes (1.5-mL microcentrifuge) Tubes (15-mL conical) Water bath (37˚C) METHOD For a general outline of the coculture procedure, see Figure 1.
Generation of RGC Reaggregates Day 1
1. Purify RGCs from three litters of P5 Sprague–Dawley rat pups by immunopanning as described in Purification and Culture of Retinal Ganglion Cells from Rodents (Winzeler and Wang 2013). 2. Resuspend the purified RGCs in 2 mL of ND-G medium. 3. Divide the RGCs (typically 2.5 million neurons) into four wells of an eight-well chamber slide. 4. Incubate the cells at 37˚C (10% CO2). Day 2
5. Gently resuspend the RGCs to promote reaggregate formation by pipetting the medium up and down eight to 12 times per well using a P200 pipette. 6. Return the cells to 37˚C (10% CO2). 7. Coat coverslips with PDL as follows.
i. Rinse 24 to 32 glass coverslips three times with sterile distilled H2O. ii. Distribute the coverslips individually around a 15-cm Petri dish. iii. Aspirate any remaining water droplets. 2 d of reaggregation
~10 d of axon growth ~
Immunopan P5 RGCs
Plate RGC reaggregates
~ ~ ~
2 h of recovery
~
~
~ ~
Immunopan OPCs from P7 optic nerves or cortex
~
~ ~
~ ~
Adenovirus infection or amaxa nucleofection
3–10 d of coculture Assess cell fate and morphology
FIGURE 1. Myelinating cocultures of OPCs with RGC reaggregates. RGCs are purified by immunopanning and allowed to reaggregate for 2 d before plating on a substrate permissive for axon growth for 7–14 d. OPCs are then acutely isolated from optic nerves or cortices and transfected if desired. Transfer of these OPCs to the reaggregate cultures initiates the coculture that results in myelination. (Reprinted, with permission, from Watkins et al. 2008.)
1082
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
iv. Dilute 100 µL of PDL stock in 10 mL of sterile distilled H2O. v. Add 100 µL of diluted PDL to the surface of each coverslip. Incubate the coverslips at room temperature for 30 min. vi. Rinse the coverslips three times with sterile distilled H2O. vii. Transfer the coverslips to the wells of 24-well culture plates. viii. Aspirate the excess H2O from each well, using the tip of the pipette to position the coverslip in the center of each well. Day 3
8. Equilibrate 6 mL of ND-G medium to 37˚C (10% CO2) in an incubator.
9. Coat the PDL-coated coverslips with laminin as follows.
i. Dilute 10 µL of mouse laminin stock into 5 mL of NB. ii. Gently add 50 µL of this diluted laminin to each coverslip from Step 7, allowing the surface tension to prevent the drop from expanding off the coverslip to the rest of the well. iii. Gently transfer the plate(s) to the incubator. iv. Incubate the plate(s) at 37˚C (10% CO2) for 4–6 h. 10. Switch the coverslips to ND-G medium as follows. i. Aspirate the laminin solution from each coverslip and immediately replace it with 30 µL of ND-G medium. ii. Return the plate(s) to the incubator. 11. Gently resuspend the RGC reaggregates in each well with a P200 pipette and transfer the contents of each well to a 1.5-mL microcentrifuge tube. 12. Wash the RGC reaggregates as follows. i. Allow the reaggregates to settle. Remove the supernatant containing debris and unaggregated RGCs. ii. Add 500 µL of ND-G medium. iii. Allow the reaggregates to settle and remove the supernatant. iv. Repeat Steps 12.ii–12.iii five to six times, or until no further debris can be seen in the supernatant under the microscope. 13. Resuspend the RGC reaggregates in 600 µL of ND-G medium. 14. Add 20 µL of medium containing reaggregates to each coverslip from Step 10. Gently distribute the reaggregates around the edge of each coverslip to avoid having the reaggregates cluster in the center. The goal is to evenly distribute the reaggregates around the surface of each coverslip. This requires gentle resuspension before plating on each coverslip, as the reaggregates tend to rapidly settle.
15. Gently return the plate(s) to the incubator. Excessive motion at this stage can result in spread of the liquid off of the coverslips or swirling of the reaggregates toward the centers of the coverslips, disrupting the even distribution.
Day 4
16. Add 450 µL of ND-G medium to each well of RGCs. 17. Feed the cultures every third day by removing 225 µL of medium and adding 250 µL of fresh equilibrated ND-G medium. See note at Step 47.
18. Seed purified OPCs on top of RGC reaggregates (Steps 46–47) after 8–12 d of culture. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1083
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
Isolation of Rat Optic Nerve OPCs by Immunopanning A schematic diagram of OPC purification is provided in Figure 2.
Day 1
19. Prepare the panning dishes by incubating three 10-cm Petri dishes overnight at 4˚C in 10 mL of 50 mM Tris (pH 9.5) plus the following secondary antibodies: i. Dish 1: goat anti-mouse IgG (H + L) (10 µg/mL) ii. Dish 2: goat anti-mouse IgG (H + L) (10 µg/mL) iii. Dish 3: goat anti-mouse IgM (µ-chain specific) (10 µg/mL)
Obtain rats and Dissect optic nerves prepare all solutions, etc. Step 28 Steps 19–27 45 min 1–2 h, day 1: 30–60 min, day 2
Cut nerves into pieces Step 29 15 min
Preparation & dissection Steps 19–29 2–3 h
Dissociation Steps 30–40 3h Papain digest tissue Steps 30–34 75–90 min
Trituration Step 35 15–30 min
Astrocytes
Rinsing cells Steps 36–40 2x 13-min spins; 45–60 min total
Oligodendrocytes
OPCs
Cell suspension
Panning Steps 41 and 42 2–2.5 h
Ran-2 panning 30 min
Trypsin digestion Steps 43.i–43.iv 6–10 min
GC panning 30 min
Squirt cells off plate Steps 43.v–43.viii 15–30 min
A2B5 panning 45 min plate + 15 min rinsing
Collect and plate cells Steps 44–47 15-min spin + 10–45 min plating
Trypsinization & plating Steps 43–47 1–2 h
FIGURE 2. Isolation of rat optic nerve OPCs by immunopanning.
1084
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
Day 2
20. Prepare a D-PBS/0.2% BSA solution by mixing 19 mL of D-PBS with 1 mL of 4% BSA stock. 21. Prepare the panning buffer as follows. i. Mix 2 mL of D-PBS/0.2% BSA with 18 mL of D-PBS. ii. Add 200 µL of insulin stock. iii. Equilibrate to room temperature. 22. Prepare the low-ovo solution as follows. i. Mix 1 mL of low-ovo stock solution with 9 mL of D-PBS. ii. Add 100 µL of 0.4% DNase stock. 23. Prepare the high-ovo solution by mixing 1.2 mL of high-ovo stock solution with 5 mL of D-PBS. 24. Add primary antibody in 0.2% D-PBS/BSA solution to the panning dishes as follows: i. Dish 1: 1 mL of RAN-2 supernatant in 4 mL of D-PBS/0.2% BSA. ii. Dish 2: 1 mL of GC supernatant in 4 mL of D-PBS/0.2% BSA. iii. Dish 3: A2B5 ascites 1:750 in D-PBS/0.2% BSA. A2B5 supernatant can also be used (at about 1:20). Immediately before adding the cell suspension to each dish, rinse with three washes of D-PBS. For mouse optic nerve OPCs, use O4 instead of A2B5 and Thy1.2 instead of RAN-2 (see Purification of Oligodendrocyte Precursor Cells from Rat Cortices by Immunopanning [Dugas and Emery 2013]).
25. Incubate the dishes at room temperature for at least 1 h before use. 26. Equilibrate 10 mL of EBSS at 37˚C (10% CO2).
27. Prepare 30% FCS as follows.
i. Mix 4.5 mL of FCS with 10.5 mL of D-PBS. ii. Filter through a 0.22-µm filter to sterilize. iii. Equilibrate to room temperature. 28. Dissect the optic nerves and optic chiasm from three litters of P7 Sprague–Dawley rat pups as follows. i. Remove the head and skull and lift the brain out. The optic nerves will be lying on the base of the skull.
ii. Cut the optic nerves behind the chiasm and also behind each eye. Grasp the chiasm with forceps. iii. Transfer the pair of nerves into a 35-mm Petri dish containing 2 mL of D-PBS. 29. Cut each nerve with fine scissors into as many pieces as possible (at least five to 10 pieces per nerve). Transfer the D-PBS and nerve pieces (excluding chiasm pieces) to a small tube. 30. Prepare a papain solution as follows. i. Add 165 U of papain to 5 mL of D-PBS. ii. Add 100 µL of 0.4% DNase stock. iii. Dissolve the papain by placing this solution in a water bath at 37˚C for a few minutes. iv. Just before use, add L-cysteine (2 mg) to activate the papain solution. v. Adjust the pH to 7.4 with 1 M NaOH. vi. Filter through a 0.22-µm filter to sterilize. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1085
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
31. Remove the D-PBS from the harvested nerve tissue with a Pasteur pipettes and add the papain solution. 32. Incubate the tissue, with occasional agitation, for 75 min in a water bath at 37˚C.
33. Remove the papain solution with a Pasteur pipette.
34. Rinse away the excess papain by adding 3 mL of low-ovo solution. Let the tissue pieces settle and remove the solution. 35. Serially triturate the nerve pieces in low-ovo solution, 1 mL at a time, with a 2.5-mL syringe and #21 gauge needle (three sets of five passes each), followed by a #23 gauge needle (three sets of five passes each), as follows. i. Add 1 mL of low-ovo solution, pass the nerve pieces through the needle one or two times, let the pieces settle, and collect the suspension above the pieces with a Pasteur pipette into a 15-mL conical tube. ii. Add another 1 mL of low-ovo solution, triturate again, and repeat as described. With this method, cells successfully removed from the nerve pieces are not repetitively passed through the needle.
36. Filter the cell suspension through an autoclaved 20-µm nylon mesh as follows. i. Form a cone with the filter on top of a 15-mL tube. ii. Prewet the filter by adding 0.5 mL of low-ovo solution. iii. Transfer the cell suspension through the filter. iv. Add the remaining volume of low-ovo solution (0.5 mL). 37. Centrifuge the dissociated cells at 200g (1000 rpm) in a clinical centrifuge for 13 min at 25˚C. Aspirate the supernatant. 38. Resuspend the cells in 1 mL of high-ovo solution, then mix in the remaining 5 mL of high-ovo solution. 39. Centrifuge for 13 min at 25˚C. Aspirate the supernatant.
If transfection is required, proceed to the section Nucleofection of Optic Nerve Cells following Step 47. Complete Steps 48–58 before continuing OPC purification at Step 40. If no transfection is needed, proceed directly to Step 40–47 to finish OPC purification.
40. Resuspend the optic nerve cells in 8 mL of panning buffer. 41. Proceed with immunopanning as follows. i. Rinse each panning dish (from Step 25) three times with D-PBS immediately before adding the cell suspension. ii. Add the cell suspension to Dish 1 (Ran 2, negative selection for astrocytes). iii. Incubate for 30 min at room temperature, agitating gently at 15 min to ensure access of all cells to the panning surface area. iv. Transfer the cell suspension to Dish 2 (GC, negative selection for OLs). v. Incubate for 20–30 min at room temperature, agitating at 10–15 min. vi. Transfer the cell suspension to Dish 3 (A2B5, positive selection for OPCs). vii. Incubate for 30 min at room temperature, agitating at 15 min. 42. Wash Dish 3 (A2B5) with D-PBS (10 mL per wash) around five times to remove nonadherent cells. Check the success of the washes under a microscope. 43. Release the purified cells from the dish as follows. i. Add 200 µL of 2.5% trypsin to 4 mL of preequilibrated EBSS. 1086
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
ii. Rinse Dish 3 (A2B5) once with the remaining 6 mL of preequilibrated EBSS. iii. Remove the EBSS wash and add the 4 mL of trypsin/EBSS. iv. Place at 37˚C (10% CO2) for 10 min. v. Add 2 mL of 30% FCS.
vi. Use a P1000 pipette to gently squirt around the surface of the dish to release the cells. vii. Collect the supernatant in a 15-mL conical tube containing 1 mL of 30% FCS. viii. Repeat Steps 43.vi and 43.vii twice, adding another 3–4 mL of 30% FCS to the dish each time. 44. Centrifuge the purified OPCs at 200g (1000 rpm) for 13 min. 45. Aspirate the supernatant carefully but completely. 46. Plate the purified OPCs onto the RGC reaggregate cultures as follows. i. Resuspend the OPCs in ND-G medium or MyM (20,000 OPCs in 0.5 mL medium). ii. For each well of RGC reaggregates, remove 300 µL of medium and add 500 µL of OPCs. Cocultures may be maintained for up to 6 d with no further manipulation.
47. For culture periods >6 d, change one-half volume of medium every 3 d. When appropriate, add the γ-secretase inhibitor DAPT to a final concentration of 1 µM to promote ensheathment. Differentiation and ensheathment occur well in either ND-G or MyM. MyM supports more reliable and robust wrapping and compaction over a short time course. MyM, however, does not support initial survival and recovery of acutely isolated RGCs as well as does ND-G. Replacement of ND-G (for purified RGC axon outgrowth) with MyM (for myelinating cocultures) is best achieved by beginning the switch with a half volume medium change in the final one or two RGC feedings before OPC isolation. The plating of OPCs as described here further reduces ND-G, and subsequent half volume feedings are sufficient to complete the changeover.
Nucleofection of Optic Nerve Cells
48. Prepare 5 mL of ND-G medium. 49. Prepare a PDL-coated tissue culture dish as follows. i. Dilute 100 µL of PDL stock in 10 mL of sterile distilled H2O. ii. Incubate a 10-cm tissue culture dish with 5 mL of PDL solution for 30 min at room temperature. iii. Rinse three times with D-PBS. 50. Make 10 mL of 20% FCS as follows. i. Mix 2 mL of FCS with 8 mL of D-PBS. ii. Filter through a 0.22-µm filter to sterilize. iii. Equilibrate to room temperature. 51. Resuspend the optic nerve cells from Step 39 in 500 µL of ND-G medium. 52. Add all the cells to the PDL-coated dish containing the remaining (4.5 mL) equilibrated ND-G medium. 53. Allow the cells to recover in the incubator for 90 min at 37˚C.
54. Release the cells from the dish as follows. i. Remove the medium and rinse the cells once with 6 mL of preequilibrated EBSS. ii. Incubate the cells at 37˚C with 4 mL of trypsin-EDTA (diluted 1:10 in EBSS) for 8 min.
iii. Remove the cells from the incubator and add 2 mL of 20% FCS. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1087
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
iv. Use a P1000 pipette to gently squirt around the surface of the dish to release the cells. v. Collect the supernatant in a 15-mL conical tube containing 1 mL of 20% FCS. vi. Repeat Steps 54.iv and 54.v twice, adding another 3–4 mL of 20% FCS each time. 55. Centrifuge the cells at 200g (1000 rpm) for 11 min. 56. Resuspend the cells in Lonza nucleofector solution. Use 105 µL of solution per transfection. 57. Perform nucleofection for each of the desired transfections as follows. i. Quickly transfer 100 µL of the cell suspension to a tube containing 1.5 µg of plasmid or siRNA. ii. Mix gently. iii. Transfer this volume to the provided cuvette. iv. Electroporate the cells using program O-17. v. Add 500 µL of ND-G to the cuvette and use the provided plastic pipette to transfer the entire volume of cells to a tube containing 8 ml of panning buffer (see Step 40). 58. Finish the isolation of optic nerve OPCs by proceeding with Steps 40–47.
RELATED INFORMATION
This protocol was adapted from Watkins et al. (2008), © 2008, with permission from Elsevier.
RECIPES Biotin Stock (5000×)
Dissolve 10 mg of biotin (Sigma-Aldrich B4639) in 200 mL of D-PBS with phenol red to prepare biotin stock. Add 0.1 N NaOH in 5-µL drops to aid in dissolving. Make 10-µL aliquots and store at −20˚C. BSA Stock (4%)
Dissolve 2 g of BSA (Sigma-Aldrich A4161) in 50 mL of D-PBS at 37˚C. Adjust pH to 7.4 with 200 µL of 1 N NaOH. Filter sequentially through 0.45-µm and 0.22-µm filters. Prepare 1.0-mL aliquots and store at −20˚C. CNTF Stock (10 μg/mL)
To prepare, dilute ciliary neurotrophic factor (CNTF; Peprotech 450-02) to 10 μg/mL with sterile 0.2% BSA in Dulbecco’s phosphate-buffered saline (D-PBS; HyClone SH30264.01). Make 20-μL aliquots, flash freeze in liquid nitrogen, and store –80˚C. Forskolin Stock (4.2 mg/mL)
To prepare, add 1 mL of sterile DMSO to a 50-mg bottle of forskolin (Sigma-Aldrich F6886) and pipette up and down until the powder is fully resuspended. Transfer to a 15-mL conical tube and add an additional 11 mL of DMSO to achieve a final concentration of 4.2 mg/mL. Store in 20- and 80-μL aliquots at −20˚C. 1088
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
High-Ovomucoid Stock (6×)
1. Dissolve the following in 160–180 mL of Dulbecco’s phosphate-buffered saline (D-PBS; Invitrogen 14287-080). Reagent
Amount (for 200 mL)
Final concentration
6g 6g
30 mg/mL 30 mg/mL
BSA (Sigma-Aldrich A-8806) Trypsin inhibitor (Worthington LS003086)
2. Adjust the pH to 7.4 with 10 N NaOH. Bring the volume to 200 mL with D-PBS, then filter-sterilize. 3. Make 1-mL aliquots, and store them at −20˚C. Hormone Mix (200×)
Reagents
Amount to add
Apotransferrin (Sigma-Aldrich T1147; 4 mg/mL in DMEM) Putrescine (Sigma-Aldrich P5780; 12.8 mg/mL in DMEM) Progesterone (Sigma-Aldrich P8783; 25 µg/µL in ethanol) Sodium selenite (Sigma-Aldrich S5261; 0.4 mg/mL in DMEM)
Final concentration
5 mL
1 mg/mL
5 mL
20 mM
10 µL
4 µM
51.9 µL
6 µM
Combine the ingredients in an additional 10 mL of DMEM. Mix well and filter through a 0.22-µm filter. Make 200-µL aliquots and store at −20˚C.
Insulin Stock (0.5 mg/mL)
To 20 mL of sterile water, add 10 mg of insulin (Sigma-Aldrich I6634) and 100 μL of 1.0 N HCl. Mix well. Filter through a 0.22-μm filter. Store at 4˚C for 4–6 wk. Low-Ovomucoid Stock Solution (10×)
To prepare, add 3 g of BSA (Sigma-Aldrich A8806) to 150 mL D-PBS. Mix well. Add 3 g of trypsin inhibitor (Worthington LS003086) and mix to dissolve. Add 1 mL of 1 N NaOH to adjust the pH to 7.4. Bring the volume to 200 mL with D-PBS. Filter-sterilize through a 0.22-μm filter. Make 1.0-mL aliquots and store at −20˚C. Myelination Medium (MyM)
1. Dilute a 10 µg/mL stock solution of CNTF to 0.1 ng/mL by combining the following: 1 µL CNTF stock (10 µg/mL) 99 µL MyM base medium 2. Add 15 µL of 0.1 ng/mL CNTF to the following: 15 mL MyM base medium 15 µLBDNF(Peprotech450-02;50 µg/mLinD-PBS[Gibco14287-080]containing0.2%BSA) 3. Equilibrate in an incubator to 37˚C/10% CO2. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1089
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
MyM Base Medium
Reagent
Amount to add (for 40 mL)
DMEM-High Glucose medium (Gibco 11960-044) Insulin stock (0.5 mg/mL) Sodium pyruvate (100 mM) (Gibco 11360-070) L-Glutamine (200 mM) (Gibco 25030-081) Hormone mix (200×) T3 stock (4 µg/mL) Hydrocortisone (50 µM) (Sigma-Aldrich H6909) Trace Elements B (1000×) (Cellgro) Biotin stock (5000×) Vitamin B12 (Sigma-Aldrich V6629; 1.36 mg/mL in D-PBS) B-27 (Invitrogen) or N21-MAX (R&D Systems) Ceruloplasmin (1 mg/mL) (Calbiochem 239799)
39 mL 400 µL 400 µL 400 µL 200 µL 200 µL 40 µL 40 µL 8 µL 8 µL 15 µL 4 µL
Filter the solution through a 0.22-µm filter to sterilize and store at 4˚C for up to 1 wk.
NAC Stock (5 mg/mL)
To prepare, dissolve 50 mg of N-acetyl-L-cysteine (NAC) powder (Sigma-Aldrich A8199) in 10 mL of Neurobasal Medium (Gibco/Life Technologies 21103). (The solution will be yellowish.) Filter through a 0.22-μm filter. Prepare 20- and 80-μL aliquots and store them at −20˚C. ND-Growth Medium (ND-G)
Reagent
Amount to add
ND-SATO base medium BDNF (Peprotech 450-02; 50 µg/mL in D-PBS containing 0.2% BSA) CNTF (10 µg/mL) Forskolin stock (4.2 mg/mL)
8 mL 8 µL 8 µL 8 µL
Equilibrate in an incubator to 37˚C/10% CO2. ND-SATO Base Medium
Reagent
Amount to add (for 40 mL)
Neurobasal solution (Gibco 21103-049) DMEM (Gibco 11960-044) Penicillin/streptomycin (100×) (Gibco 15140-122) L-Glutamine (200 mM) (Gibco 25030-081) SATO (100×) Sodium pyruvate (100 mM) (Gibco 11360-070) Insulin stock (0.5 mg/mL) T3 stock (4 µg/mL) NAC stock (5 mg/mL) Trace Elements B (1000×) (Cellgro) Biotin stock (5000×) B-27 (Invitrogen) or N21-MAX (R&D Systems)
19.5 mL 19.5 mL 400 µL 400 µL 400 µL 400 µL 400 µL 400 µL 40 µL 40 µL 8 µL 800 µL
Filter the solution through a 0.22-µm filter to sterilize and store at 4˚C for up to 1 wk. 1090
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
SATO (100×)
1. Prepare the following stock solutions (these should be made fresh; do not reuse). • Combine 2.5 mg of progesterone (Sigma-Aldrich P8783) and 100 µL of ethanol to make a progesterone stock solution. • Combine 4.0 mg of sodium selenite (Sigma-Aldrich S5261), 10 µL of 1 N NaOH, and 10 mL of Neurobasal (NB, Gibco 21103-049) to make a sodium selenite stock solution. 2. Add the following to 40 mL of DMEM (Gibco 11960-044): Reagent
Quantity
Final concentration in medium (1×)
BSA (Sigma-Aldrich A4161) Transferrin (Sigma-Aldrich T1147) Putrescine dihydrochloride (SigmaAldrich P5780) Progesterone stock solution Sodium selenite stock solution
400 mg 400 mg 64 mg
100 µg/mL 100 µg/mL 16 µg/mL
10 µL 400 µL
60 ng/mL 40 ng/mL
3. Mix well, and filter-sterilize through a prerinsed 0.22-µm filter. Make 200-µL aliquots, and store at −20˚C. T3 Stock (4 μg/mL)
Dissolve 3.2 mg of 3,3′ ,5-triiodo-L-thyronine sodium salt (T3; Sigma-Aldrich T6397) in 400 µL of 0.1 N NaOH. Add 10 µL of T3 solution to 20 mL of Dulbecco’s phosphatebuffered saline (D-PBS; Gibco 14190-144). Filter through a 0.22-µm filter, discarding the first 10 mL. Make 200-µL aliquots, and store at −20˚C.
REFERENCES Dugas JC, Emery B. 2013. Purification of oligodendrocyte precursor cells from rat cortices by immunopanning. Cold Spring Harb Protoc doi: 10.1101/pdb.prot070862. Watkins TA, Emery B, Mulinyawe S, Barres BA. 2008. Distinct stages of myelination regulated by γ-secretase and astrocytes in a rapidly myelinating CNS coculture system. Neuron 60: 555–569.
Winzeler A, Wang JT. 2013. Purification and culture of retinal ganglion cells from rodents. Cold Spring Harb Protoc doi: 10.1101/pdb .prot074906.
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1091
Myelinating Cocultures of Rat Retinal Ganglion Cell Reaggregates and Optic Nerve Oligodendrocyte Precursor Cells Trent A. Watkins and Anja R. Scholze Cold Spring Harb Protoc; doi: 10.1101/pdb.prot074971 Email Alerting Service Subject Categories
Receive free email alerts when new articles cite this article - click here. Browse articles on similar topics from Cold Spring Harbor Protocols. Immunoaffinity Purification (34 articles) Immunoseparation (21 articles) Mouse (307 articles) Neural Cell Culture (52 articles) Neuroscience, general (255 articles) Other Laboratory Organisms (56 articles)
To subscribe to Cold Spring Harbor Protocols go to:
http://cshprotocols.cshlp.org/subscriptions
© 2014 Cold Spring Harbor Laboratory Press
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Protocol
Myelinating Cocultures of Rat Retinal Ganglion Cell Reaggregates and Optic Nerve Oligodendrocyte Precursor Cells Trent A. Watkins1,2,3 and Anja R. Scholze1 1
Department of Neurobiology, Stanford University School of Medicine, Stanford, California 94305-5125
This protocol describes the generation of a rapidly myelinating central nervous system coculture for the study of complex neuronal-glial interactions in vitro. Postnatal rat retinal ganglion cells (RGCs) purified by immunopanning are promoted to cluster into reaggregates and then allowed to extend dense beds of radial axons for 10–14 d. Subsequently, rodent oligodendrocyte precursor cells are purified by immunopanning, transfected if desired, and seeded on top of the RGC reaggregates. Under the conditions described here, compact myelin can be observed within 6 d.
MATERIALS It is essential that you consult the appropriate Material Safety Data Sheets and your institution’s Environmental Health and Safety Office for proper handling of equipment and hazardous material used in this protocol. RECIPES: Please see the end of this article for recipes indicated by . Additional recipes can be found online at http://cshprotocols.cshlp.org/site/recipes.
Reagents
BSA stock (4%) DNase To prepare a 0.4% stock of DNase in Earle’s balanced salt solution (EBSS), add 1 mL of Earle’s balanced salt solution (EBSS; Sigma-Aldrich E6267) per 12,500 U of DNase I (Worthington LS002007). Keep on ice. Filter sterilize, and store in 200-μL aliquots at −20˚C.
Dulbecco’s phosphate-buffered saline (D-PBS, Gibco 14287-080) Earle’s balanced salt solution (EBSS) (Sigma-Aldrich E6267) Fetal calf serum (FCS) (Gibco 10437-028) Prepare 50-mL aliquots of FCS. Heat-inactivate aliquots for 30 min at 55˚C, and then store at −20˚C.
High-ovomucoid (high-ovo) stock solution (6×) Immunopanning reagents A2B5 hybridoma supernatant or ascites Galactocerebroside (GC) hybridoma supernatant Goat anti-mouse IgG + IgM (H + L) (Jackson ImmunoResearch 115-005-044)
2
Present address: Department of Neuroscience, Genentech Inc., South San Francisco, California 94080. Correspondence: [email protected]
3
© 2014 Cold Spring Harbor Laboratory Press Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1080
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
Goat anti-mouse IgM, μ-chain specific (Jackson ImmunoResearch 115-005-020) Ran-2 hybridoma supernatant
InSolution γ-Secretase Inhibitor I× (DAPT) (Calbiochem/EMD Millipore 565784) (optional; see Step 47) Insulin stock (0.5 mg/mL) L-Cysteine hydrochloride monohydrate (Sigma-Aldrich C7880) Laminin (mouse) (Cultrex; R&D Systems 3400-010-01) Thaw mouse laminin (1 mg/mL) at 4˚C. Make 10-µL aliquots and store at −30˚C.
Low-ovomucoid (low-ovo) stock solution Myelination medium (MyM), freshly prepared NaOH (1 M) ND-SATO growth medium (ND-G), freshly prepared Neurobasal solution (NB) (Gibco 21103-049) Nucleofector kit for rat glial cells (Lonza) (if performing transfection) Papain (Worthington LS03126) Plasmid or siRNA for transfection, as desired Poly-D-lysine (PDL) (1 mg/mL) Rat pups (P5, Sprague–Dawley) for purification of RGCs (three litters/30 animals) P4–P10 pups may be used, but P5 is optimal for its combination of yield, viability, and purity. RGCs are purified using the method described in Purification and Culture of Retinal Ganglion Cells from Rodents (Winzeler and Wang 2013). Mouse RGCs can also be used, per the provided modifications.
Rat pups (P7, Sprague–Dawley) for purification of optic nerve OPCs (3 litters/30 animals) Pups of nearly any postnatal age will work for OPC purification, but P6–P8 produces the best yield. Cortical OPCs can also be used in the myelinating cocultures and the OPC yields are much higher (2–3 million per P7 cortex; see Purification of Oligodendrocyte Precursor Cells from Rat Cortices by Immunopanning [Dugas and Emery 2013]). Mouse OPCs can also be used, per the provided modifications.
Tris-HCl (50 mM, pH 9.5; sterile) Dissolve 12.1 g of Trizma base in 200 mL of dH2O. Adjust pH to 9.5 with HCl.
Trypsin stock (2.5%) Dissolve trypsin (Sigma-Aldrich T9935) at 30,000 U/mL in EBSS (Sigma-Aldrich E6267). Filter through a 0.22-µm filter. Make 200-µL aliquots and store at −80˚C.
Trypsin-EDTA (Gibco) (if performing transfection) Equipment
Centrifuge (tabletop, with 15- and 50-mL conical tube adaptors) Centrifuge (clinical) Chamber slides (eight well) Coverslips (glass) We wash our glass coverslips with at least three washes of 70% ethanol over 7 d on a rotating platform and store in 70% ethanol.
Dissection tools Forceps Scissors (fine) Scissors (sharp)
Incubator (37˚C/10% CO2) Microscope Needles (#21 and #23 gauge) Nylon mesh filter (20 µm) (Nitex; Tetko) Nucleofector I device (Lonza) (if performing transfection) Pasteur pipettes Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1081
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
Petri dishes (35, 10, 15 cm) Syringes (2.5 mL) Syringe filters (0.22 µm) Tissue culture dishes (10 cm) (if performing transfection) Tissue culture plates (24-well) Tubes (1.5-mL microcentrifuge) Tubes (15-mL conical) Water bath (37˚C) METHOD For a general outline of the coculture procedure, see Figure 1.
Generation of RGC Reaggregates Day 1
1. Purify RGCs from three litters of P5 Sprague–Dawley rat pups by immunopanning as described in Purification and Culture of Retinal Ganglion Cells from Rodents (Winzeler and Wang 2013). 2. Resuspend the purified RGCs in 2 mL of ND-G medium. 3. Divide the RGCs (typically 2.5 million neurons) into four wells of an eight-well chamber slide. 4. Incubate the cells at 37˚C (10% CO2). Day 2
5. Gently resuspend the RGCs to promote reaggregate formation by pipetting the medium up and down eight to 12 times per well using a P200 pipette. 6. Return the cells to 37˚C (10% CO2). 7. Coat coverslips with PDL as follows.
i. Rinse 24 to 32 glass coverslips three times with sterile distilled H2O. ii. Distribute the coverslips individually around a 15-cm Petri dish. iii. Aspirate any remaining water droplets. 2 d of reaggregation
~10 d of axon growth ~
Immunopan P5 RGCs
Plate RGC reaggregates
~ ~ ~
2 h of recovery
~
~
~ ~
Immunopan OPCs from P7 optic nerves or cortex
~
~ ~
~ ~
Adenovirus infection or amaxa nucleofection
3–10 d of coculture Assess cell fate and morphology
FIGURE 1. Myelinating cocultures of OPCs with RGC reaggregates. RGCs are purified by immunopanning and allowed to reaggregate for 2 d before plating on a substrate permissive for axon growth for 7–14 d. OPCs are then acutely isolated from optic nerves or cortices and transfected if desired. Transfer of these OPCs to the reaggregate cultures initiates the coculture that results in myelination. (Reprinted, with permission, from Watkins et al. 2008.)
1082
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
iv. Dilute 100 µL of PDL stock in 10 mL of sterile distilled H2O. v. Add 100 µL of diluted PDL to the surface of each coverslip. Incubate the coverslips at room temperature for 30 min. vi. Rinse the coverslips three times with sterile distilled H2O. vii. Transfer the coverslips to the wells of 24-well culture plates. viii. Aspirate the excess H2O from each well, using the tip of the pipette to position the coverslip in the center of each well. Day 3
8. Equilibrate 6 mL of ND-G medium to 37˚C (10% CO2) in an incubator.
9. Coat the PDL-coated coverslips with laminin as follows.
i. Dilute 10 µL of mouse laminin stock into 5 mL of NB. ii. Gently add 50 µL of this diluted laminin to each coverslip from Step 7, allowing the surface tension to prevent the drop from expanding off the coverslip to the rest of the well. iii. Gently transfer the plate(s) to the incubator. iv. Incubate the plate(s) at 37˚C (10% CO2) for 4–6 h. 10. Switch the coverslips to ND-G medium as follows. i. Aspirate the laminin solution from each coverslip and immediately replace it with 30 µL of ND-G medium. ii. Return the plate(s) to the incubator. 11. Gently resuspend the RGC reaggregates in each well with a P200 pipette and transfer the contents of each well to a 1.5-mL microcentrifuge tube. 12. Wash the RGC reaggregates as follows. i. Allow the reaggregates to settle. Remove the supernatant containing debris and unaggregated RGCs. ii. Add 500 µL of ND-G medium. iii. Allow the reaggregates to settle and remove the supernatant. iv. Repeat Steps 12.ii–12.iii five to six times, or until no further debris can be seen in the supernatant under the microscope. 13. Resuspend the RGC reaggregates in 600 µL of ND-G medium. 14. Add 20 µL of medium containing reaggregates to each coverslip from Step 10. Gently distribute the reaggregates around the edge of each coverslip to avoid having the reaggregates cluster in the center. The goal is to evenly distribute the reaggregates around the surface of each coverslip. This requires gentle resuspension before plating on each coverslip, as the reaggregates tend to rapidly settle.
15. Gently return the plate(s) to the incubator. Excessive motion at this stage can result in spread of the liquid off of the coverslips or swirling of the reaggregates toward the centers of the coverslips, disrupting the even distribution.
Day 4
16. Add 450 µL of ND-G medium to each well of RGCs. 17. Feed the cultures every third day by removing 225 µL of medium and adding 250 µL of fresh equilibrated ND-G medium. See note at Step 47.
18. Seed purified OPCs on top of RGC reaggregates (Steps 46–47) after 8–12 d of culture. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1083
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
Isolation of Rat Optic Nerve OPCs by Immunopanning A schematic diagram of OPC purification is provided in Figure 2.
Day 1
19. Prepare the panning dishes by incubating three 10-cm Petri dishes overnight at 4˚C in 10 mL of 50 mM Tris (pH 9.5) plus the following secondary antibodies: i. Dish 1: goat anti-mouse IgG (H + L) (10 µg/mL) ii. Dish 2: goat anti-mouse IgG (H + L) (10 µg/mL) iii. Dish 3: goat anti-mouse IgM (µ-chain specific) (10 µg/mL)
Obtain rats and Dissect optic nerves prepare all solutions, etc. Step 28 Steps 19–27 45 min 1–2 h, day 1: 30–60 min, day 2
Cut nerves into pieces Step 29 15 min
Preparation & dissection Steps 19–29 2–3 h
Dissociation Steps 30–40 3h Papain digest tissue Steps 30–34 75–90 min
Trituration Step 35 15–30 min
Astrocytes
Rinsing cells Steps 36–40 2x 13-min spins; 45–60 min total
Oligodendrocytes
OPCs
Cell suspension
Panning Steps 41 and 42 2–2.5 h
Ran-2 panning 30 min
Trypsin digestion Steps 43.i–43.iv 6–10 min
GC panning 30 min
Squirt cells off plate Steps 43.v–43.viii 15–30 min
A2B5 panning 45 min plate + 15 min rinsing
Collect and plate cells Steps 44–47 15-min spin + 10–45 min plating
Trypsinization & plating Steps 43–47 1–2 h
FIGURE 2. Isolation of rat optic nerve OPCs by immunopanning.
1084
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
Day 2
20. Prepare a D-PBS/0.2% BSA solution by mixing 19 mL of D-PBS with 1 mL of 4% BSA stock. 21. Prepare the panning buffer as follows. i. Mix 2 mL of D-PBS/0.2% BSA with 18 mL of D-PBS. ii. Add 200 µL of insulin stock. iii. Equilibrate to room temperature. 22. Prepare the low-ovo solution as follows. i. Mix 1 mL of low-ovo stock solution with 9 mL of D-PBS. ii. Add 100 µL of 0.4% DNase stock. 23. Prepare the high-ovo solution by mixing 1.2 mL of high-ovo stock solution with 5 mL of D-PBS. 24. Add primary antibody in 0.2% D-PBS/BSA solution to the panning dishes as follows: i. Dish 1: 1 mL of RAN-2 supernatant in 4 mL of D-PBS/0.2% BSA. ii. Dish 2: 1 mL of GC supernatant in 4 mL of D-PBS/0.2% BSA. iii. Dish 3: A2B5 ascites 1:750 in D-PBS/0.2% BSA. A2B5 supernatant can also be used (at about 1:20). Immediately before adding the cell suspension to each dish, rinse with three washes of D-PBS. For mouse optic nerve OPCs, use O4 instead of A2B5 and Thy1.2 instead of RAN-2 (see Purification of Oligodendrocyte Precursor Cells from Rat Cortices by Immunopanning [Dugas and Emery 2013]).
25. Incubate the dishes at room temperature for at least 1 h before use. 26. Equilibrate 10 mL of EBSS at 37˚C (10% CO2).
27. Prepare 30% FCS as follows.
i. Mix 4.5 mL of FCS with 10.5 mL of D-PBS. ii. Filter through a 0.22-µm filter to sterilize. iii. Equilibrate to room temperature. 28. Dissect the optic nerves and optic chiasm from three litters of P7 Sprague–Dawley rat pups as follows. i. Remove the head and skull and lift the brain out. The optic nerves will be lying on the base of the skull.
ii. Cut the optic nerves behind the chiasm and also behind each eye. Grasp the chiasm with forceps. iii. Transfer the pair of nerves into a 35-mm Petri dish containing 2 mL of D-PBS. 29. Cut each nerve with fine scissors into as many pieces as possible (at least five to 10 pieces per nerve). Transfer the D-PBS and nerve pieces (excluding chiasm pieces) to a small tube. 30. Prepare a papain solution as follows. i. Add 165 U of papain to 5 mL of D-PBS. ii. Add 100 µL of 0.4% DNase stock. iii. Dissolve the papain by placing this solution in a water bath at 37˚C for a few minutes. iv. Just before use, add L-cysteine (2 mg) to activate the papain solution. v. Adjust the pH to 7.4 with 1 M NaOH. vi. Filter through a 0.22-µm filter to sterilize. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1085
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
31. Remove the D-PBS from the harvested nerve tissue with a Pasteur pipettes and add the papain solution. 32. Incubate the tissue, with occasional agitation, for 75 min in a water bath at 37˚C.
33. Remove the papain solution with a Pasteur pipette.
34. Rinse away the excess papain by adding 3 mL of low-ovo solution. Let the tissue pieces settle and remove the solution. 35. Serially triturate the nerve pieces in low-ovo solution, 1 mL at a time, with a 2.5-mL syringe and #21 gauge needle (three sets of five passes each), followed by a #23 gauge needle (three sets of five passes each), as follows. i. Add 1 mL of low-ovo solution, pass the nerve pieces through the needle one or two times, let the pieces settle, and collect the suspension above the pieces with a Pasteur pipette into a 15-mL conical tube. ii. Add another 1 mL of low-ovo solution, triturate again, and repeat as described. With this method, cells successfully removed from the nerve pieces are not repetitively passed through the needle.
36. Filter the cell suspension through an autoclaved 20-µm nylon mesh as follows. i. Form a cone with the filter on top of a 15-mL tube. ii. Prewet the filter by adding 0.5 mL of low-ovo solution. iii. Transfer the cell suspension through the filter. iv. Add the remaining volume of low-ovo solution (0.5 mL). 37. Centrifuge the dissociated cells at 200g (1000 rpm) in a clinical centrifuge for 13 min at 25˚C. Aspirate the supernatant. 38. Resuspend the cells in 1 mL of high-ovo solution, then mix in the remaining 5 mL of high-ovo solution. 39. Centrifuge for 13 min at 25˚C. Aspirate the supernatant.
If transfection is required, proceed to the section Nucleofection of Optic Nerve Cells following Step 47. Complete Steps 48–58 before continuing OPC purification at Step 40. If no transfection is needed, proceed directly to Step 40–47 to finish OPC purification.
40. Resuspend the optic nerve cells in 8 mL of panning buffer. 41. Proceed with immunopanning as follows. i. Rinse each panning dish (from Step 25) three times with D-PBS immediately before adding the cell suspension. ii. Add the cell suspension to Dish 1 (Ran 2, negative selection for astrocytes). iii. Incubate for 30 min at room temperature, agitating gently at 15 min to ensure access of all cells to the panning surface area. iv. Transfer the cell suspension to Dish 2 (GC, negative selection for OLs). v. Incubate for 20–30 min at room temperature, agitating at 10–15 min. vi. Transfer the cell suspension to Dish 3 (A2B5, positive selection for OPCs). vii. Incubate for 30 min at room temperature, agitating at 15 min. 42. Wash Dish 3 (A2B5) with D-PBS (10 mL per wash) around five times to remove nonadherent cells. Check the success of the washes under a microscope. 43. Release the purified cells from the dish as follows. i. Add 200 µL of 2.5% trypsin to 4 mL of preequilibrated EBSS. 1086
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
ii. Rinse Dish 3 (A2B5) once with the remaining 6 mL of preequilibrated EBSS. iii. Remove the EBSS wash and add the 4 mL of trypsin/EBSS. iv. Place at 37˚C (10% CO2) for 10 min. v. Add 2 mL of 30% FCS.
vi. Use a P1000 pipette to gently squirt around the surface of the dish to release the cells. vii. Collect the supernatant in a 15-mL conical tube containing 1 mL of 30% FCS. viii. Repeat Steps 43.vi and 43.vii twice, adding another 3–4 mL of 30% FCS to the dish each time. 44. Centrifuge the purified OPCs at 200g (1000 rpm) for 13 min. 45. Aspirate the supernatant carefully but completely. 46. Plate the purified OPCs onto the RGC reaggregate cultures as follows. i. Resuspend the OPCs in ND-G medium or MyM (20,000 OPCs in 0.5 mL medium). ii. For each well of RGC reaggregates, remove 300 µL of medium and add 500 µL of OPCs. Cocultures may be maintained for up to 6 d with no further manipulation.
47. For culture periods >6 d, change one-half volume of medium every 3 d. When appropriate, add the γ-secretase inhibitor DAPT to a final concentration of 1 µM to promote ensheathment. Differentiation and ensheathment occur well in either ND-G or MyM. MyM supports more reliable and robust wrapping and compaction over a short time course. MyM, however, does not support initial survival and recovery of acutely isolated RGCs as well as does ND-G. Replacement of ND-G (for purified RGC axon outgrowth) with MyM (for myelinating cocultures) is best achieved by beginning the switch with a half volume medium change in the final one or two RGC feedings before OPC isolation. The plating of OPCs as described here further reduces ND-G, and subsequent half volume feedings are sufficient to complete the changeover.
Nucleofection of Optic Nerve Cells
48. Prepare 5 mL of ND-G medium. 49. Prepare a PDL-coated tissue culture dish as follows. i. Dilute 100 µL of PDL stock in 10 mL of sterile distilled H2O. ii. Incubate a 10-cm tissue culture dish with 5 mL of PDL solution for 30 min at room temperature. iii. Rinse three times with D-PBS. 50. Make 10 mL of 20% FCS as follows. i. Mix 2 mL of FCS with 8 mL of D-PBS. ii. Filter through a 0.22-µm filter to sterilize. iii. Equilibrate to room temperature. 51. Resuspend the optic nerve cells from Step 39 in 500 µL of ND-G medium. 52. Add all the cells to the PDL-coated dish containing the remaining (4.5 mL) equilibrated ND-G medium. 53. Allow the cells to recover in the incubator for 90 min at 37˚C.
54. Release the cells from the dish as follows. i. Remove the medium and rinse the cells once with 6 mL of preequilibrated EBSS. ii. Incubate the cells at 37˚C with 4 mL of trypsin-EDTA (diluted 1:10 in EBSS) for 8 min.
iii. Remove the cells from the incubator and add 2 mL of 20% FCS. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1087
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
iv. Use a P1000 pipette to gently squirt around the surface of the dish to release the cells. v. Collect the supernatant in a 15-mL conical tube containing 1 mL of 20% FCS. vi. Repeat Steps 54.iv and 54.v twice, adding another 3–4 mL of 20% FCS each time. 55. Centrifuge the cells at 200g (1000 rpm) for 11 min. 56. Resuspend the cells in Lonza nucleofector solution. Use 105 µL of solution per transfection. 57. Perform nucleofection for each of the desired transfections as follows. i. Quickly transfer 100 µL of the cell suspension to a tube containing 1.5 µg of plasmid or siRNA. ii. Mix gently. iii. Transfer this volume to the provided cuvette. iv. Electroporate the cells using program O-17. v. Add 500 µL of ND-G to the cuvette and use the provided plastic pipette to transfer the entire volume of cells to a tube containing 8 ml of panning buffer (see Step 40). 58. Finish the isolation of optic nerve OPCs by proceeding with Steps 40–47.
RELATED INFORMATION
This protocol was adapted from Watkins et al. (2008), © 2008, with permission from Elsevier.
RECIPES Biotin Stock (5000×)
Dissolve 10 mg of biotin (Sigma-Aldrich B4639) in 200 mL of D-PBS with phenol red to prepare biotin stock. Add 0.1 N NaOH in 5-µL drops to aid in dissolving. Make 10-µL aliquots and store at −20˚C. BSA Stock (4%)
Dissolve 2 g of BSA (Sigma-Aldrich A4161) in 50 mL of D-PBS at 37˚C. Adjust pH to 7.4 with 200 µL of 1 N NaOH. Filter sequentially through 0.45-µm and 0.22-µm filters. Prepare 1.0-mL aliquots and store at −20˚C. CNTF Stock (10 μg/mL)
To prepare, dilute ciliary neurotrophic factor (CNTF; Peprotech 450-02) to 10 μg/mL with sterile 0.2% BSA in Dulbecco’s phosphate-buffered saline (D-PBS; HyClone SH30264.01). Make 20-μL aliquots, flash freeze in liquid nitrogen, and store –80˚C. Forskolin Stock (4.2 mg/mL)
To prepare, add 1 mL of sterile DMSO to a 50-mg bottle of forskolin (Sigma-Aldrich F6886) and pipette up and down until the powder is fully resuspended. Transfer to a 15-mL conical tube and add an additional 11 mL of DMSO to achieve a final concentration of 4.2 mg/mL. Store in 20- and 80-μL aliquots at −20˚C. 1088
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
High-Ovomucoid Stock (6×)
1. Dissolve the following in 160–180 mL of Dulbecco’s phosphate-buffered saline (D-PBS; Invitrogen 14287-080). Reagent
Amount (for 200 mL)
Final concentration
6g 6g
30 mg/mL 30 mg/mL
BSA (Sigma-Aldrich A-8806) Trypsin inhibitor (Worthington LS003086)
2. Adjust the pH to 7.4 with 10 N NaOH. Bring the volume to 200 mL with D-PBS, then filter-sterilize. 3. Make 1-mL aliquots, and store them at −20˚C. Hormone Mix (200×)
Reagents
Amount to add
Apotransferrin (Sigma-Aldrich T1147; 4 mg/mL in DMEM) Putrescine (Sigma-Aldrich P5780; 12.8 mg/mL in DMEM) Progesterone (Sigma-Aldrich P8783; 25 µg/µL in ethanol) Sodium selenite (Sigma-Aldrich S5261; 0.4 mg/mL in DMEM)
Final concentration
5 mL
1 mg/mL
5 mL
20 mM
10 µL
4 µM
51.9 µL
6 µM
Combine the ingredients in an additional 10 mL of DMEM. Mix well and filter through a 0.22-µm filter. Make 200-µL aliquots and store at −20˚C.
Insulin Stock (0.5 mg/mL)
To 20 mL of sterile water, add 10 mg of insulin (Sigma-Aldrich I6634) and 100 μL of 1.0 N HCl. Mix well. Filter through a 0.22-μm filter. Store at 4˚C for 4–6 wk. Low-Ovomucoid Stock Solution (10×)
To prepare, add 3 g of BSA (Sigma-Aldrich A8806) to 150 mL D-PBS. Mix well. Add 3 g of trypsin inhibitor (Worthington LS003086) and mix to dissolve. Add 1 mL of 1 N NaOH to adjust the pH to 7.4. Bring the volume to 200 mL with D-PBS. Filter-sterilize through a 0.22-μm filter. Make 1.0-mL aliquots and store at −20˚C. Myelination Medium (MyM)
1. Dilute a 10 µg/mL stock solution of CNTF to 0.1 ng/mL by combining the following: 1 µL CNTF stock (10 µg/mL) 99 µL MyM base medium 2. Add 15 µL of 0.1 ng/mL CNTF to the following: 15 mL MyM base medium 15 µLBDNF(Peprotech450-02;50 µg/mLinD-PBS[Gibco14287-080]containing0.2%BSA) 3. Equilibrate in an incubator to 37˚C/10% CO2. Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1089
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
T.A. Watkins and A.R. Scholze
MyM Base Medium
Reagent
Amount to add (for 40 mL)
DMEM-High Glucose medium (Gibco 11960-044) Insulin stock (0.5 mg/mL) Sodium pyruvate (100 mM) (Gibco 11360-070) L-Glutamine (200 mM) (Gibco 25030-081) Hormone mix (200×) T3 stock (4 µg/mL) Hydrocortisone (50 µM) (Sigma-Aldrich H6909) Trace Elements B (1000×) (Cellgro) Biotin stock (5000×) Vitamin B12 (Sigma-Aldrich V6629; 1.36 mg/mL in D-PBS) B-27 (Invitrogen) or N21-MAX (R&D Systems) Ceruloplasmin (1 mg/mL) (Calbiochem 239799)
39 mL 400 µL 400 µL 400 µL 200 µL 200 µL 40 µL 40 µL 8 µL 8 µL 15 µL 4 µL
Filter the solution through a 0.22-µm filter to sterilize and store at 4˚C for up to 1 wk.
NAC Stock (5 mg/mL)
To prepare, dissolve 50 mg of N-acetyl-L-cysteine (NAC) powder (Sigma-Aldrich A8199) in 10 mL of Neurobasal Medium (Gibco/Life Technologies 21103). (The solution will be yellowish.) Filter through a 0.22-μm filter. Prepare 20- and 80-μL aliquots and store them at −20˚C. ND-Growth Medium (ND-G)
Reagent
Amount to add
ND-SATO base medium BDNF (Peprotech 450-02; 50 µg/mL in D-PBS containing 0.2% BSA) CNTF (10 µg/mL) Forskolin stock (4.2 mg/mL)
8 mL 8 µL 8 µL 8 µL
Equilibrate in an incubator to 37˚C/10% CO2. ND-SATO Base Medium
Reagent
Amount to add (for 40 mL)
Neurobasal solution (Gibco 21103-049) DMEM (Gibco 11960-044) Penicillin/streptomycin (100×) (Gibco 15140-122) L-Glutamine (200 mM) (Gibco 25030-081) SATO (100×) Sodium pyruvate (100 mM) (Gibco 11360-070) Insulin stock (0.5 mg/mL) T3 stock (4 µg/mL) NAC stock (5 mg/mL) Trace Elements B (1000×) (Cellgro) Biotin stock (5000×) B-27 (Invitrogen) or N21-MAX (R&D Systems)
19.5 mL 19.5 mL 400 µL 400 µL 400 µL 400 µL 400 µL 400 µL 40 µL 40 µL 8 µL 800 µL
Filter the solution through a 0.22-µm filter to sterilize and store at 4˚C for up to 1 wk. 1090
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
Downloaded from http://cshprotocols.cshlp.org/ at COLUMBIA UNIVERSITY on October 1, 2014 - Published by Cold Spring Harbor Laboratory Press
Myelinating Cocultures of RGCs and OPCs
SATO (100×)
1. Prepare the following stock solutions (these should be made fresh; do not reuse). • Combine 2.5 mg of progesterone (Sigma-Aldrich P8783) and 100 µL of ethanol to make a progesterone stock solution. • Combine 4.0 mg of sodium selenite (Sigma-Aldrich S5261), 10 µL of 1 N NaOH, and 10 mL of Neurobasal (NB, Gibco 21103-049) to make a sodium selenite stock solution. 2. Add the following to 40 mL of DMEM (Gibco 11960-044): Reagent
Quantity
Final concentration in medium (1×)
BSA (Sigma-Aldrich A4161) Transferrin (Sigma-Aldrich T1147) Putrescine dihydrochloride (SigmaAldrich P5780) Progesterone stock solution Sodium selenite stock solution
400 mg 400 mg 64 mg
100 µg/mL 100 µg/mL 16 µg/mL
10 µL 400 µL
60 ng/mL 40 ng/mL
3. Mix well, and filter-sterilize through a prerinsed 0.22-µm filter. Make 200-µL aliquots, and store at −20˚C. T3 Stock (4 μg/mL)
Dissolve 3.2 mg of 3,3′ ,5-triiodo-L-thyronine sodium salt (T3; Sigma-Aldrich T6397) in 400 µL of 0.1 N NaOH. Add 10 µL of T3 solution to 20 mL of Dulbecco’s phosphatebuffered saline (D-PBS; Gibco 14190-144). Filter through a 0.22-µm filter, discarding the first 10 mL. Make 200-µL aliquots, and store at −20˚C.
REFERENCES Dugas JC, Emery B. 2013. Purification of oligodendrocyte precursor cells from rat cortices by immunopanning. Cold Spring Harb Protoc doi: 10.1101/pdb.prot070862. Watkins TA, Emery B, Mulinyawe S, Barres BA. 2008. Distinct stages of myelination regulated by γ-secretase and astrocytes in a rapidly myelinating CNS coculture system. Neuron 60: 555–569.
Winzeler A, Wang JT. 2013. Purification and culture of retinal ganglion cells from rodents. Cold Spring Harb Protoc doi: 10.1101/pdb .prot074906.
Cite this protocol as Cold Spring Harb Protoc; 2014; doi:10.1101/pdb.prot074971
1091
